Bioscience Explained | Vol 1, No 1

bioscience | explained Vol 1 | No 1

Dean Madden NCBE, The University of Reading Britt-Marie Lidesten Erik Dahlbergsgymnasiet, Jönköping

Bacterial illumination Culturing luminous bacteria

Passengers on board cruise liners are sometimes startled at night by an eerie glow emitted by seawater flush lavatories. The explanation lies in luminous microorganisms, which require oxygen and sometimes physical damage for their light-generating reactions. Such microbes are found in all marine environments [1]. Their presence can be a problem — much of the research on luminescent dinoflagellates has been funded by the US Navy, embarrassed by glowing trails behind ships, and more recently by interference with the laser light used for submarine communications [2]. Luminous bacteria can be very useful, however. They are highly sensitive to pollution and are often used to detect toxins in water, often in place of animal tests [3]. The enzyme and reactants that produce light are also used for analytical work in vitro [4]. The bacterium Photobacterium phosphoreum (Figure 1) is the brightest of all luminescent bacteria. It is able to thrive at room CORRESPONDENCE TO temperatures (20–25°C or lower), so it does not need specialist Dean Madden incubation facilities. In addition, the bacterium’s requirement for National Centre for Biotechnology saline conditions means that it is unlikely to survive for long if Education, The University of Reading, accidentally spilt; hence it is often recommended for elementary Whiteknights, Reading, RG6 6AP school work [5]. The United Kingdom. P. phosphoreum may simply be cultivated to learn aseptic techniques. [email protected] Individual McCartney bottles of broth are a convenient means of culturing the organism, although greater volumes in a well-aerated Britt-Marie Lidesten fermenter look particularly spectacular! More challenging, open- Erik Dahlbergsgymnasiet, ended practical investigations are also possible. Föreningsgatan 2, 550 02 Jönköping Sweden. If time and local safety regulations allow, luminous bacteria can be [email protected] isolated from the skin of seafish or squid using an enriched medium.

Fig. 1 Photobacterium phosphoreum streaked onto a Petri dish. This photograph was taken using a long exposure. To the naked eye, glowing cultures of this organism generally appear more green than blue. Strong luminescence like this can only be achieved using a special Photobacterium growth medium.

Fig. 2 How do bacteria make light? Luciferase from Photobacterium phosphoreum. Data for this computer The light-emitting reaction is a side branch of the electron transport model was obtained from the Protein chain of bacterial respiration. Unlike light production in dinoﬂagellates, Data Bank, ID code 1FVP. See Reference [6]. which only luminesce in response to physical stress, bacterial luminescence is a continuous process. In the reaction, reduced luciferin

(FMNH2) forms a complex with the enzyme luciferase. This complex reacts with molecular oxygen to form oxidised luciferin (oxyluciferin or FMN) and ‘excited’ luciferase. Long-chain aldehydes are also required in this stage of the reaction. The return of luciferase to its resting state results in the emission of a photon (Figure 5).

Why do bacteria make light? Although its biochemistry is well-understood, for many years the function of bacterial luminescence remained a mystery. The amount of light generated by an individual cell is too small to be of physiological or ecological significance. It is now known that free-living bacteria in the sea do not produce luciferase and consequently do not emit light. It is only when such cells are packed together, for example, in the specialised light organs of a fish or squid, that they glow brightly. In Fig. 3 this symbiotic relationship, the animal provides the bacteria with a The structure of bacterial luciferin. nutrient-rich environment for growth. In return, the bacterial light gives the animal a means of attracting prey, communication, or O camouflage [1]. CH2 (CHOH)3 CH2 O POH Why do bacteria glow inside light organs and not in the open sea? H N N O O Laboratory observations in the late 1960s showed that newly- H NH N inoculated cultures of Vibrio fischeri only begin to emit light once the H cell density has reached a certain level (Figure 4). At first it was suggested that this was because the culture media contained an inhibitor of the light-generating reaction, which was removed by the bacteria when greater numbers were present. Later this was shown to be wrong. In fact, the accumulation of an activator molecule or ‘autoinducer’ at high cell densities is responsible for triggering the Fig. 4 production of light. The bacteria are able to sense cell density by The development of luminescence and detecting not only the presence, but also the concentration of the the production of luciferase (measured autoinducer. Today this type of cell-to-cell signalling is called quorum by its cross-reaction with antibodies) sensing [for a brief history, see Reference 8]. compared with the growth of Vibrio fischeri. See Reference [7]. The basic mechanisms of quorum sensing identified in V. fischeri and Vibrio harveyi are now known to be similar to those which 100 Growth optical density at 660 nm regulate gene expression in a wide variety of Gram-negative species. Luciferase cross-reacting material Quorum sensing is also found in Gram-positive bacteria and there is Luminescence in vitro even evidence that communication occurs between species (‘quorum Luminescence in vivo 10 sensing cross talk’). Bacterial communication like this is now thought to play a major role in the virulence of pathogens and in enzyme and antibiotic production. Research in this field could therefore be of considerable economic and medical importance. 1.0 In conjunction with work on quorum sensing, the regulation of bacterial luminescence at the genetic level has been studied [9, 10]. There are seven so-called lux genes that are required to produce and 0.1 regulate luminescence in V. fischeri. Two plasmids containing these genes have been constructed for educational use, and their function can be demonstrated by inserting them into Escherichia coli [11]. 0.01 IMPORTANT! Within the European Union, such bacterial 12345 transformation experiments may only be undertaken with the Time/Hours permission of the appropriate authorities (in the UK, the HSE [12]).

Fig. 5 The reactions thought to be involved in the generation of light by bacteria. These are a offshoot of the electron transport chain.

NADH2 NAD

FMN FMNH2 Luciferase

FMNH2 complex Luciferase (RESTING STATE)

LIGHT O2 R-COH (460–490 nm)

H2O R-COOH

FMN a ﬂavin mononucleotide (luciferin)

EXCITED R-CHO FMN Luciferase long-chain aldehyde

Aim To observe luminescence from Photobacterium phosphoreum or Vibrio ﬁscheri.

Equipment and materials

Needed by each person or group • Slope culture of P. phosphoreum or V. ﬁscheri • Photobacterium or seawater broth (see recipes below). For small- scale cultivation by individuals, dispense the broth in aliquots of 15 ml into McCartney bottles before autoclaving. • Bunsen burner • Inoculation loop • Access to a dark room for viewing the cultures 18–24 hours after inoculation

Procedure 1 Inoculate the broth and incubate overnight at room temperature (20–25 °C). Aeration is necessary for luminescence. This can be achieved by shaking the McCartney bottles — but they must be tightly-capped. IMPORTANT! Sufﬁcient space must be left above the liquid in the bottles, to permit the production of gas and to allow oxygen to diffuse into the culture.

Safety Good microbiological practice must be observed by anyone carrying out this work. The method for isolation of luminous bacteria from ﬁsh (see box, page 6) may not be appropriate for use in schools. If this work is undertaken, the precautions described must be followed to ensure that pathogens are not inadvertently isolated and cultivated. For further guidance, teachers in the United Kingdom should consult Topics in Safety [5] and ensure that they abide by any additional guidelines set by their local authority or school governing body.

Media recipes

Photobacterium medium Seawater medium Artiﬁcial seawater • Seawater aquarium salt 33 g • Yeast extract 10 g • NaCl 28.13 g • Yeast extract 5 g • Malt extract 4 g • KCl 0.77 g

• Tryptone 5 g • Glucose 4 g • CaCl2.2H2O 1.60 g

• Glycerol 3 g • Seawater* 750 ml • MgCl2.6H2O 4.80 g

• Tris 6 g • Distilled water 250 ml • NaHCO3 0.11 g

•NH4Cl 5 g • MgSO4.7H2O 3.50 g • Distilled water 1 l • Distilled water 1 l For a solid medium add 15–20 g agar. Adjust to pH 7.5. For a solid medium add: * Natural sea water must be stored • CaCO 1 g 3 in the dark for at least three weeks All culture media should be • Agar 15–20 g to ‘age’. If natural sea water is not autoclaved at 121°C for Adjust to pH 7.2–7.5. available use artiﬁcial sea water. 15–20 minutes before use.

Preparation Slope cultures of P. phosphoreum are able to survive prolonged storage at 4 °C. However, such cultures will need to be ‘revived’ by transferring them onto fresh slopes or plates before they are suitable use by a class. This should be done 2–3 days before they are required. McCartney bottles of broth may be prepared and autoclaved in advance and kept until they are needed.

Timing Only actively-growing cultures will emit light. Maximum luminescence usually develops 18–24 hours after inoculation.

Troubleshooting Cultures must be grown on Photobacterium medium if bright illumination is to be observed. Only cultures in the ‘log phase’ of growth will produce light (see Figure 4).

Photography Photographs of plates or ﬂasks inoculated with luminous bacteria can be taken using monochrome 125 ISO ﬁlm at f/2.0 for 100 seconds. The light produced by small volumes of cultures cannot usually be detected by a digital still or video camera, although it might be possible to photograph greater volumes using such equipment.

Further investigations

1 Effect of temperature on luminescence Cultures in McCartney bottles may be placed in water baths at a range of temperatures (e.g., 0–60 °C) to investigate the effect of temperature on generation of light. The proteins required for luminescence in P. phosphoreum and V. fischeri are denatured at 37 °C, and whereas P. phosphoreum will grow at 4 °C, V. fischeri will not. Remember that bioluminescence is an enzyme-catalysed process. Unlike fluorescence (e.g., of green fluorescent protein, GFP) a product does not accumulate inside the cells, so luminescence will only be seen in living cultures.

2 Effect of oxygen concentration Oxygen is required for light-generation, hence in a tube of bacterial culture that has been left to stand, luminescence will only be observed at the interface of the air and the liquid. Methylene blue solution is blue when oxidised and colourless when reduced. It may therefore be used to monitor oxygen concentration within a culture. To do this, add 0.5 ml of 0.01% aqueous methylene blue to a 15 ml culture of luminous bacteria in a McCartney bottle. Allow the tube to stand for about 20 minutes, or until luminescence is only observed at the top of the liquid. It will be noticed that light production is restricted to the blue, oxygenated zone while in the liquid beneath, where bacterial respiration has depleted the oxygen, the methylene blue is colourless.

3 Antibiotic action Vibrio is Gram-negative and P. phosphoreum and V. ﬁscheri are sensitive to a range of antibiotics. This sensitivity may be tested by placing paper discs (e.g., Oxoid Multodiscs) that have been impregnated with antibiotic onto the surface of plates spread with bacteria. Dark zones around the discs indicate areas where bacteria have not grown.

4 Effect of pollutants P. phosphoreum is highly-sensitive to environmental pollutants and is often used to detect their presence. The effect of metal ions on luminesence can be investigated. Suitable metal ions and concentrations include: lead (1 mg/l); copper (1 mg/l); cobalt (3 mg/l) or manganese (5 mg/l). A spectacular demonstration of the effect of chemicals such as bleach may be performed by adding a few drops to a culture of luminescing bacteria. The ‘lights go out’ almost immediately!

Isolation of luminous bacteria from fish or squid P. phosphoreum is one of the commonest spoilage organisms of fish such Fish enrichment medium as cod. It is not known to cause disease, but there are reports of people • Boil 250 g fish meat in 1 l of being startled by glowing fish fingers in the fridge! water. To isolate glowing bacteria from fish, obtain a freshly-caught seafish or • Add 30 g NaCl and sieve to squid. It is very important that the fish has not been frozen or washed in remove solids. fresh water. It is also better if the fish has not been stored on ice. Place • Add 10 g peptone, 10 ml the fish in a container with 3% NaCl solution. The liquid should be deep glycerol and 1 g yeast extract. enough to come half way up the fish. • Adjust the pH to 7. Cover the container and store the fish for 24 hours at about 12–15 °C. • Autoclave at 121 °C for 15–20 Note: if this temperature is difficult to achieve, place the fish in a fridge minutes. at about 4 °C for 48–72 hours. After incubation, take the container with the fish into a dark room. For a solid medium add 15–20 g of When your eyes have been adopted to dark, light spots will be visible on agar to every litre of broth. the skin of the fish. Use a sterile toothpick or disposable sterile loop or needle to aseptically transfer the brightest spots onto sterile fish enrichment agar plates. Tip: some people find it useful to use a dim red lamp (e.g., a photographic safety lamp) for this step. Turn the fish away Safety note from the lamp so that the glowing colonies are in the shade and therefore visible. Several species of Vibrio are pathogenic. The chances of Transfer the cultures to new agar plates every second day if you are inadvertently isolating pathogens incubating them at temperatures around 12 °C or once a week if you are in this procedure can be reduced storing them in a fridge. P. phosphoreum will grow at 4 °C; V. fischeri will by using at least 3% salt solution not. By selecting the brightest colonies when inoculating, it should be and incubating fish and plates at possible to isolate a pure culture. no more than 15 °C. Human pathogens are unlikely to grow under such conditions.

Suppliers Lyophilised cultures of Photobacterium phosphoreum may be obtained from culture collections such as the UK National Collections of Industrial, Marine and Food Bacteria or the German Collection of Microorganisms and Cells. Several school science suppliers (e.g., Blades Biological) provide suitable slope cultures at lower cost. A practical kit for cultivating P. phosphoreum is available from Philip Harris Education in the UK (‘Bioluminescence kit’, Cat. No. A02264); Carolina Biological in the USA sells a kit for cultivating V. ﬁscheri (‘Bioluminescent bacterium kit’, Cat. No. BA-15-4750).

References 1 Widder, E.A. (2001) Marine bioluminescence. Why do so many animals in the open ocean make light? Bioscience Explained 1 (1) http://www.bioscience-explained.org/EN1.1 2 US Naval Oceanographic Office. Bioluminescence Survey Systems http://www.navo.navy.mil/biolum/blwebpge.htm 3 Azur Environmental Toxicity Testing http://www.azurenv.com 4 Cambridge Research and Technology Transfer Limited http://www.lumiweb.com 5 Topics in safety (Third edition) (2001) Hatfield: Association for Science Education. ISBN: 0 863 57316 9. 6 Kita, A., Kasai, S. and Miki, K. (1995) Crystal structure determination of a flavoprotein FP390 from a luminescent bacterium, Photobacterium phosphoreum. Journal of Biochemistry (Tokyo) 117, 575. 7 Nealson, K.H., Platt, T. and Hastings, J.W. (1970) Cellular control of the synthesis and activity of the bacterial luminescence system. Journal of Bacteriology 104, 313–322. 8 Hastings, J.W. and Greenberg, E.P. (1999) Quorum sensing: the explanation of a curious phenomenon reveals a common characteristic of bacteria. Journal of Bacteriology 181 (8) 2667–2668. 9 Slock, J. (1995) The lux system of bioluminescence, or, how to ‘sense’ your neighbor. The American Biology Teacher 57 (4) 222–224. 10 Bluth, B.J., Frew, S.E. and McNally, B. (1997) Cell-cell communication and the lux operon in Vibrio fischeri http://www.bio.cmu.edu/ courses/03441/TermPapers 97TermPapers/lux/default.html 11 Slock, J. (1995) Transformation experiment using bioluminescence genes of Vibrio fischeri. The American Biology Teacher 57 (4) 225–227. 12 A guide to the genetically-modified organisms (Contained Use) Regulations 2000. Health and Safety Executive (2000) London: The Stationery Office. ISBN: 0 7176 1758 0.

Further reading Turley, C.M. (1982) ‘An illuminating demonstration of bacterial luminescence’. In A sourcebook of experiments for the teaching of microbiology (eds. Primrose, S.B. and Wardlaw, A.C.) pp. 11–15. London: Academic Press. ISBN: 0 12 565680 7. This book is out-of- print, but a PDF ﬁle of the article can be downloaded from here. Salmond, G.P.C. et al (1995) The bacterial ‘enigma’: cracking the code of cell–cell communication. Molecular Microbiology 16 (4) 615–624. Losick, R. and Kaiser, D. (1997) Why and how bacteria communicate. Scientiﬁc American 276 (2) 68–73.

Web sites Luxgene.com — Glow-in-the-dark organisms http://www.luxgene.com Luminescent bacteria http://www.biology.pl/bakterie_sw/index_en.html HBOI — More about bioluminescence http://www.biolum.org/ Marine bioluminescence page http://lifesci.ucsb.edu/~biolum/ Scripps bioluminescence page http://siobiolum.ucsd.edu/Biolum_intro.html

Acknowledgements Thanks to Pat C. Hickey of the Fungal Cell Biology Group at the University of Edinburgh for the photograph of Photobacterium phosphoreum. The cartoon (Figure 5) was originally drawn by Paul Stevens for the NCBE Newsletter in 1990. John Richardson of the Scottish Schools Equipment Research Centre and John Grainger of The University of Reading advised on the safety and practicality of the bacterial isolation procedure. Enevold Fahlsen from Göteborg University also gave expert advice about safety questions. The Society for General Microbiology kindly permitted the reproduction of C.M. Turley’s 1982 educational article on bacterial luminescence (see link in ‘Further reading’).